Is it possible that among just three recognized markers specifically targeting DNA, none exists beyond the microbial world? Researchers, spearheaded by Xiaoqi Feng from ISTA, have now shown that this particular marker, N4-methylcytosine (4mC), plays a crucial role in sperm development and maturation within the liverwort species Marchantia polymorpha, an important model organism in studying plant evolution.
The findings are published in Cell .
Occasionally, it is a living fossil that leads to some of the most intriguing discoveries regarding life and evolution. As a cousin of mosses, the liverwort known as Marchantia represents one of the earliest surviving plant species and likely belongs to the oldest lineages of plants to inhabit terrestrial environments.
Besides its key role in helping plants make the shift from aquatic to terrestrial environments, Marchantia continues to employ an ancient method of reproduction that has largely disappeared in most plant species.
While other organisms have developed forms of sexual reproduction not dependent on water, Marchantia sperm relies on droplets of rainfall for release and subsequent swimming to reach and fertilize neighboring females. Nevertheless, numerous details regarding how Marchantia sperm functions at a molecular level remain unclear even today.
The team headed by Xiaoqi Feng at the Institute of Science and Technology Austria (ISTA) employs this distinctive plant as a model to reveal previously unidentified molecular mechanisms that provide fresh insights into the development of sexual reproduction.
Our research offers definitive proof of a novel DNA marker in organisms such as plants or animals that plays a crucial role—it is vital for sexual reproduction in Marchantia, particularly influencing sperm formation in males," explains Feng. These discoveries might be applicable in biotechnology, offering potential pathways for modulating gene activity without changing the fundamental DNA structure.
An extensive exploration past the domain of microorganisms
N4-methylcytosine (4mC) serves as an immune mechanism in bacteria, protecting their genome from degradation by incorporating a methyl chemical group at specific sites, effectively disguising them from destructive enzymes. This modification is part of the trio of recognized DNA markers which includes 4mC, 5mC, and 6mA. These chemical tags do not alter the fundamental genetic sequence but instead overlay it; hence they are termed "epigenetic markers." The term derives from the Greek prefix 'epi,' signifying "above," "beyond," or "around."
Although both 4mC and 5mC represent chemical alterations of the cytosine base within DNA—a fundamental component of this genetic material—6mA uniquely affects the adenine residue. Unlike 6mA, which lacked definitive evidence of occurrence in plant or animal species, 4mC has long captivated researchers' attention.
The stages are 'insane'—but essential for agile sperm
Feng and her team sought to unravel the molecular processes underlying sperm function in Marchantia. They identified two significant phases of widespread DNA methylation throughout sperm development. Essentially, these two stages encompass the sperm’s genetic material completely.
They demonstrated that the initial wave was linked to 5mC, a DNA modification recognized in both animals and plants, which specifically targets and suppresses what are known as "jumping genes."
However, 5mC alone could not explain the second wave of extensive methylation, which targets short sequences of two nucleotides scattered all over coding genes, the so-called CG (cytosine-guanine) dinucleotides.
In addition, the team showed that genes with a sequence similarity to N4-cytosine methyltransferases, the enzymes that catalyze 4mC methylation in bacteria, were expressed in the same window of the plant's sperm development. Could the team just have stumbled upon the enzymes for 4mC outside of microbes?
Inspired by this finding, they employed numerous quantitative methods to demonstrate that 4mC was truly the cause. Their research revealed that 4mC accounted for approximately 15% of methylated cytosines in the fully developed Marchantia sperm, compared to just under 1% in bacteria.
“By employing a comprehensive set of definitive experimental methods, we can confidently assert the validity of our findings,” states Feng. “The quantities of 4mC found in Marchantia sperm are astonishing to consider.”
Thus, the team demonstrated that the extensive 5mC and 4mC methylation waves were orchestrated during the plant's sperm development. In addition, they showed that these "crazy" levels of 4mC had key functions in the Marchantia sperm.
Without 4mC, the plant's male gametes lose their ability to effectively compete for fertilization; they move more slowly and with reduced directionality. Their overall fertility significantly declines as well. Even when fertilization does occur occasionally, the initial stages of embryo development tend to be impaired.
Horizontal gene transfer
A different type of DNA methylation known as 6mA was mistakenly reported by various researchers across multiple species prior to discovering that these findings were actually due to bacterial contamination. This error has tainted the perception among scientists and resulted in heightened oversight within the study of DNA methylation.
"Using several independent techniques for detecting DNA methylation is crucial, and we aimed to ensure our findings were robust. However, the substantial amount of 4mC actually bolstered our assurance because bacteria do not typically possess such extensive methylation levels," explains Feng.
However, how did 4mC originate in Marchantia initially? The explanation can be found in the exchange of genetic information among distinct species without involving sexual reproduction, which is an evolutionary process known as horizontal gene transfer, or HGT.
HGT occurs commonly—at least over long periods of evolution—between bacteria and plants. It is likely through this mechanism that 4mC made its way into Marchantia. These transfer events involving soil bacteria to plants played a crucial role in facilitating the shift of plants from water-based to land-dwelling habitats.
"Perhaps this is an example of one of nature’s spontaneous occurrences that accelerated evolutionary processes. The transfer of genetic information from bacteria to Marchantia proved beneficial for the plant, leading to retention and selection of this characteristic," says Feng.
More mC awaiting discovery?
So far, the liverwort remains the sole plant genus wherein 4mC has definitively been observed. "Our observations likely aren’t an accident," remarks Feng. Indeed, during initial development phases in mammals, significant remodifications typically occur as substantial portions of DNA’s epigenetic markers get erased and then newly established.
Therefore, we may have to examine a particular phase of growth in other organisms. We could find 4mC present as well, just awaiting discovery.
This research illuminates an essential aspect of sexual reproduction that has developed in nature and might have practical uses in biotechnology. Through examining the exact role of the 4mC epigenetic alteration in Marchantia, this molecular process could possibly serve as a technique for epigenetic genome manipulation.
More information: Comprehensive N4 Cytosine Methylation Is Crucial for Marchantia Sperm Function Cell (2025). DOI: 10.1016/j.cell.2025.03.014 . www.cell.com/cell/fulltext/S0092-8674(25)00287-9
Furnished by the Institute of Science and Technology Austria
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